Tuberculosis (TB) is the most frequent cause of death from a single infectious agent. While active disease results from about 5% of exposures, most develop a 'latent' infection without symptoms. Most cases of recurrent TB result from 'reactivation' of such infections, although exogenous re-infection is a significant factor in endemic regions, calling into question the efficacy of 'naturally-acquired' immunity. Live Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the only vaccine currently available. While protective in children, it has had negligible impact on the global Tb epidemic and is unsafe in immunodeficient individuals. We have recently shown that consecutive immunization with DNA vaccines and attenuated fowlpoxvirus (FPV) vectors encoding similar model vaccine antigens generates high levels of antigen-specific, interferon-gamma-secreting CD4+ and CD8+ T cells that persist in vivo for at least 6 months and, importantly, exhibit markedly increased sensitivity for the immunizing antigen. In terms of T cell memory, antigen challenge leads to rapid expansion of systemic and mucosal T cell effectors in DNA/FPV prime-boosted animals, reaching levels as high as 30% of total T cell numbers. The latter may be particularly important in pulmonary TB, where little attention has been paid to local immune responses. The primary aim of this proposal is test the hypothesis that prime-boost vaccination-induced T cell responses will control primary infection and reactivation of latent TB infection. Our goal is to generate protective T cell responses in both systemic and mucosal (pulmonary) tissues against key mycobacterial antigens expressed in acute and latent stages of infection. Specifically, we will determine protective efficacy of systemic (IM) prime-boosting against (i) acute infection following low-close aerosol challenge of vaccinated mice with M. tuberculosis and (ii) reactivation in a murine model of latent TB disease. We will also (iii) study the protective capacity of our prime-boost vaccines when given mucosally (IN) and will characterize systemic and mucosal (pulmonary) CD4+ and CD8+ T cell responses in each of these models. Codon optimization of mycobacterial genes expressed in our vaccines and co-delivery of IL-15 genes will be used in attempts to further enhance the magnitude and memory of T cell responses. The capacity of this approach to generate strong, sustained Th1-type CD4+ and CD8+ T cell responses is particularly attractive in the context of TB and HIV infection, where maintenance of Th1 responses is critical for protective immunity.